Partition
Liquid Chromatography
Principles of Separation
Liquid-Liquid Extraction
Liquid-liquid extraction:
Partition of a solute between two nonmiscible liquid phases
Partition coefficient
K 
C sup
C inf
To totally extract the desired solute
K must be either very large or very small
Principles of Separation
Partition Chromatography
Static representation (dynamic in reality)
Zone 1 Zone 2 Zone 3
Zone n
Mobile phase
Solute molecules
Stationary phase
Each solute partitions in the two phases according to its own K
= according to its relative affinity for the two phases
K 
C stationary
C mobile
Principles of Separation
Interactions
Solute
SP
MP
Principles of Separation
Partition Chromatography
The equilibrium is respected in the whole column
Each zone is called a « theoretical plate »
K must not be too large otherwise the retention is too long
(too much affinity for the stationary phase)
K 
C stationary
C mobile
K must be large enough for the solutes to be a little retained
in the stationary phase otherwise no separation is possible
(too much affinity for the mobile phase)
For a separation to occur: K ≠ K
If K > K,
remains longer in the column than
Principles of Separation
Retention of the solute depends on:
- The nature of the solute
- The nature of the stationary and mobile phases
- The relative affinity of the solute for SP and MP
- The specific surface area of the stationary phase (more SP = more interactions)
- The temperature (can influence the thermodynamic equilibrium K)
Retention does not depend on:
- The geometrical parameters of the column (length, internal diameter…)
- The particle diameter of the SP
- The flow rate
- The amount of solute injected
Principles of Separation
Separation depends on:
- The nature of the solutes
- The nature of the stationary and mobile phases
- The relative affinity of the solutes for SP and MP
- The specific surface area of the stationary phase (more SP = more interactions)
- The temperature (can influence the thermodynamic equilibria K1 and K2)
Separation does not depend on:
- The geometrical parameters of the column (length, internal diameter…)
- The particle diameter of the SP
- The flow rate
- The amount of solute injected
Principles of Separation
SP is a liquid
Separation is based on relative
solubilities in MP and SP
Normal Phase-partition was first described
but Reversed Phase-partition is now more common
NP
RP
Polar Stationary Phase
Non Polar Mobile Phase
Non Polar Stationary Phase
Polar Mobile Phase
NPLC-Partitioning
Polar interactions
Acidic
HO
HO
Dipole
O
N≡C
Basic
H
N
H
Si
H
H
O
Si
O
Si
H
O
O
Si O
Si
O
O
CYANO
Si
O
O
Si
H
Si
DIOL
Si
O
O
O
O
H
O
Si
O
AMINO
Si
O
RPLC-Partitioning
Polymers: PS-DVB
Hydrophobic interactions
Alkyl chains
from C1 to
C30
London
forces
London forces
+
π-π interactions
Si
H
H
O
Si
O
Si
H
O
O
Si O
Si
C8
O
O
Si
O
O
Si
H
Si
C18
Si
O
O
O
O
H
O
Si
O
PHENYL
Si
O
RPLC-Partitioning
OctaDecyl-Siloxane phases (ODS)
A great variety of stationary phases
None of them is identical to the other!!
Example ODS phases
PEG 300
PEG 300, 24.61% (w/w) methanol on different C18 columns
Trathnigg et al., J. Chromatogr. A, 1128 (2006) 39-44
Partitioning
First step is to determine which mode to use, NP or RP?
If sample is water insoluble or non polar, use NP
If sample is water soluble or not soluble but polar, use RP
MP is never a single solvent, always a blend of two or more
hexane
CCl4
THF
acetonitrile
methanol
Optimum polarity is obtained by mixing solvents
water
Mobile phase
Not all solvents are usable
MeOH, ACN, THF, H2O are the most widely used in RPLC
HXN, CH2Cl2, iPrOH are the most widely used in NPLC
All are low viscosity
available in high purity
not too expensive
UV transparent
miscible in each other
Mobile phase
Determining the optimum RP solvent blend
Start with a single solvent and water
Adjust the % of water from 0% on up until the best separation is obtained
(optimum k for peaks of interest)
Create blends using each of the other solvents and water that have the same
polarity
Evaluate each solvent for improvements in peak shape or movement of
selective peaks
A mix of any of the blended solvents is then evaluated for optimum resolution
Possible elution gradient (similar to T gradient in GC)
Mobile phase
elution gradient
-Total analysis time is reduced
- overall resolution is improved
- better peak shapes are possible
- improved sensitivity
Requires a compatible detector
Total flow rate is held constant
Only the proportion of the solvents are changed
Example: carbohydrate analysis, NPLC
CH2OH
O
H
H
H
OH
H
OH
OH
CH2OH
CH2OH
H
OH
glucose
O
H
O
H
OH
OH
H
H
H
H
OH
O
H
OH
H
H
OH
OH
Maltose = di-glucose
CH2OH
CH2OH
CH2OH
H
CH2OH
O
O
H
H
OH
O
H
H
H
OH
CH2OH
O
H
OH
H
O
OH
H
O
H
OH
OH
OH
OH
H
OH
H
H
H
OH
OH
Maltotriose
fructose
oligosaccharides
H
OH
Example: carbohydrate analysis, NPLC
Carbohydrates in beer
Typical chromatogram for
separation of five carbohydrates
(1) fructose
(2) glucose
(3) maltose
(4) maltotriose
(5) maltotetraose
Spherisorb NH2 (250 x 4.6 mm, 5 μm)
ACN-H2O gradient elution, 1 mL/min
Detection: ELSD
Beer sample
Noguetra et al., J. Chromatogr. A, 1065 (2005) 207-210
Example: homologous series, RPLC
15 homologous n-alkylbenzenes, linear gradient of MeOH in H2O
on ODS
P. Jandera, J. Chromatogr. A, 845 (1999) 133-144
Example: homologous series, RPLC
30 homologous oligostyrenes, linear gradient of dioxane in n-heptane
on silica gel
P. Jandera, J. Chromatogr. A, 845 (1999) 133-144
Example: anti-diabetic sulfamide drugs
Pharmaceutical conterfeiting
RPLC chromatograms of
standard mixture,
Pingtangan capsules,
Zhiwuyidaosu capsules,
Gliclazide tablets
Alltima C18 (150 x 4.6 mm, 5 μm)
MeOH- pH 3 phosphate buffer
(70:30), 1 mL/min
UV-detection
1
2
3
4
5
Yao et al., J. Chromatogr. B, 2007 (in press)
6
LC-MS coupling
Studied from 1974
Most important difficulties:

Important quantity of solvent to eliminate

Fragile molecules

Wide range of polarity and molecular weight of analytes
LC-MS coupling
Electrospray Ionisation (ESI)
+ - - + +
- +- -+ + + Capillary
3-5 kV
Drop
containing
the ions
++
-- +
+-+-+-+
+
-+-+-+-- +
+
During the evaporation of the
solvent, th electric field inside the
drop increases and ejects the ions
(electrostatic repulsion)
LC-MS coupling
Atmospheric Pressure Chemical Ionisation (APCI)
Solutes
Solute Ions [M+H]+
Solvent Molecules
Heated Nebuliser
are formed
N2
+
Liquid
+
+
N2
+
+
+
Formation
of an aerosol
Vaporisation of
the solvent and
sample
+
Charge transfer
and collision
Corona needle
Solvent molecules are ionised
LC-MS coupling
Atmospheric Pressure Chemical Ionisation (APCI)
Electrospray ( ESI )
1.000
EI / CI
Molecular Weight
100.000
Non-Polar
APCI
Polar
Example LC-UV / MS: penicillin in urine
100
%
UV 268nm
13
100
MS - SIM
m/z 333 + m/z 349
%
0
1.00
2.00
3.00
4.00
5.00
6.00
Time
Example LC-ESI-MS: vitamins analysis
LC/ESI-SIR
chromatogram of a
0.2 mg/L standard
mixture.
(1) Taurine
(2) nicotinic acid
(3) Nicotinamide
(4) pantothenic acid
(5) pyridoxal;
(6) Pyridoxine
(7) hippuric acid
(8) Thiamine
(9) Biotin
(10)Riboflavin
(11)ascorbic acid
(12)folic acid
Chen et al., Analytica Chimica Acta, 569 (2006) 169-175
Example LC-ESI-MS: vitamins analysis
(1) Taurine
(2) nicotinic acid
(3) Nicotinamide
(4) pantothenic acid
(5) pyridoxal;
(6) Pyridoxine
(7) hippuric acid
(8) Thiamine
(9) Biotin
(10)Riboflavin
(11)ascorbic acid
(12)folic acid
Example LC-ESI-MS: vitamins analysis
LC/ESI-SIR
chromatogram of a
multivitamin tablet
sample.
(1) Taurine
(2) nicotinic acid
(3) Nicotinamide
(4) pantothenic acid
(5) pyridoxal;
(6) Pyridoxine
(7) hippuric acid
(8) Thiamine
(9) Biotin
(10)Riboflavin
(11)ascorbic acid
(12)folic acid
Chen et al., Analytica Chimica Acta, 569 (2006) 169-175
Example LC-APCI-MS: Phenolic acids in industrial wastewater
Example LC-APCI-MS: Phenolic acids in industrial wastewater
Column: Porous Graphitic Carbon (10 x 2.1 mm, 5 μm)
Mobile Phase: Gradient elution with A- MeOH-ACN-Formic acid 0.2M (40:40:20)
and B- THF, flow rate 1 mL/min
Detection : APCI, negative mode
Organic molecules are "embraced" by the carbon chains of the stationary phase
Unlike the typical organic target molecule peptides and
proteins adsorb to the stationary phase often by multi-point attachment
In contrast to bulk water, hydrophobic surfaces are
covered by a
shell of highly ordered water molecules.
The chromatograms show the effect of varying the organic solvent concentration
in isocratic experiments. Note that no concentration is capable of eluting all four
components in the same run
Classical" hydrophobic organic molecules are sensitive to the carbon
chain length, while more or less identical results are obtained for
proteins and larger peptides, regardless of the carbon chain length
This picture shows hydrophobic and hydrophilic
parts on the surface of lysozyme.
The most hydrophobic parts are dark red,
the less hydrophobic lighter red.
The most hydrophilic parts are shown in dark
blue,
while the less hydrophilic parts are lighter blue
Purification by partitioning the sample
between two liquid phases.
The distribution is controlled by the
difference in polar properties of the
respective phases
Reversed Phase Chromatography utilises
solubility differences between
the sample components by a continuous
re-partitioning mechanism
The adsorption of hydrophobic molecules
is a reversible reaction whose equilibrium
is controlled by the salt concentration
The desorption curve is
shifted to the right with
increasing net hydrophobicity
Partitioning
Stationary phase
Bonded silica
Polar ligands
Normal-Phase LC
« Classical Partition Chromatography »
Non polar ligands
Reversed-Phase LC
Between NP and RP, the elution order will be somewhat reversed but not exactly,
other factors must be considered
Mixed stationary phases
May have both bonded ligands
Partitioning
In RP, polarity of the solvent determines how long the solutes are retained (k)
Snyder classed solvents according to acidic, basic, dipole characters
Proton acceptor
MeOH
THF
H2O
Proton donor
dipole
Partitioning
Temperature
T has a minimal effect on the separation
It allows obtaining thiner peaks = higher resolution
High temperatures require resistant columns
Particular vs. monolithic